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PMID:1761539

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Citation

Segal, DM and Eichler, DC (1991) A nucleolar 2'-O-methyltransferase. Specificity and evidence for its role in the methylation of mouse 28 S precursor ribosomal RNA. J. Biol. Chem. 266:24385-9

Abstract

Methylation of ribose moieties appears to be an essential post-transcriptional event in ribosomal RNA maturation. Although the sites of ribose methylation have been identified, the components involved in the 2'-O-methylation of precursor ribosomal RNA in mammalian cells have not yet been elucidated. To investigate the involvement of a recently isolated nucleolar 2'-O-methyltransferase in this process, an in vitro synthesized 28 S rRNA transcript containing a unique tandem triple 2'-O-methylated ribose site was used as a substrate. Activity assays demonstrated that this transcript served as a substrate for the nucleolar 2'-O-methyltransferase. The distribution of incorporated methyl groups was determined by hydrolyzing the 2'-O-methylated transcript with RNase followed by chromatography of the digested products on an anion-exchange high performance liquid chromatography column. Results showed one unique RNase-resistant 2'-O-methylated product, a tetramer. The position of the tetrameric sequence in the 28 S rRNA transcript was determined using RNase protection analysis which mapped the methylations to a 20-nucleotide region spanning the unique tandem triple 2'-O-methylated ribose site in 28 S rRNA. To confirm the absolute specificity of methylation, direct sequence analysis was carried out on the tandem triple 2'-O-methylated tetramer. The sequence determined for the tetramer, AmGmCmA, corresponded exactly with that reported from in vivo studies. These findings demonstrate that the purified nucleolar 2'-O-methyltransferase can accurately methylate at a specific site of an in vitro derived preribosomal RNA transcript and support the proposed involvement of this nucleolar enzyme in ribosomal RNA maturation.

Links

PubMed

Keywords

Animals; Base Sequence; Cell Nucleolus/metabolism; Chromatography, High Pressure Liquid; Methylation; Methyltransferases/metabolism; Mice; Molecular Sequence Data; RNA Precursors/metabolism; RNA Processing, Post-Transcriptional; RNA, Ribosomal, 28S/metabolism; Substrate Specificity; Transcription, Genetic

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

MOUSE:MRM2

involved_in

GO:0031167: rRNA methylation

ECO:0000303: author statement without traceable support used in manual assertion

P

Seeded From UniProt

complete

MOUSE:MRM2

involved_in

GO:0006364: rRNA processing

ECO:0000303: author statement without traceable support used in manual assertion

P

Seeded From UniProt

complete


See also

References

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